Immunotherapy is emerging as a new pillar of cancer treatment with potential to cure. However, many patients still fail to respond to these therapies. Among the underlying factors, an immunosuppressive tumor microenvironment (TME) plays a major role. Here we show that monocyte-mediated gene delivery of IFNα inhibits leukemia in a mouse model. IFN gene therapy counteracts leukemia-induced expansion of immunosuppressive myeloid cells and imposes an immunostimulatory program to the TME, as shown by bulk and single-cell transcriptome analyses. This reprogramming promotes T-cell priming and effector function against multiple surrogate tumor-specific antigens, inhibiting leukemia growth in our experimental model. Durable responses are observed in a fraction of mice and are further increased combining gene therapy with checkpoint blockers. Furthermore, IFN gene therapy strongly enhances anti-tumor activity of adoptively transferred T cells engineered with tumor-specific TCR or CAR, overcoming suppressive signals in the leukemia TME. These findings warrant further investigations on the potential development of our gene therapy strategy towards clinical testing. © 2018, The Author(s).